Use of vanadium compounds to accelerate bone healing

ABSTRACT

This invention provides a method of promoting bone healing by locally administering a vanadium-based insulin mimetic agent to a patient in need thereof. The invention also provides a new use of insulin-mimetic vanadium compounds for manufacture of medicaments for accelerating bone-healing processes. In addition, the invention also encompasses a bone injury treatment kit suitable for localized administration of insulin-mimetic vanadium compounds or compositions thereof to a patient in need of such treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/295,234, filed on Jan. 15,2010, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel method for bone healing inpatients by local administration of insulin-mimetic vanadium compoundsas anabolic agents. The invention encompasses methods for treatment ofbone injuries with insulin-mimetic vanadium compounds or compositionsthereof and use of insulin-mimetic vanadium compounds for manufacture ofmedicaments for treatment of bone injuries, as well as bone injurytreatment kits suitable for local administration of insulin-mimeticvanadium compounds.

BACKGROUND OF THE INVENTION

About six million bone fractures, including about 600,000 non-unioncases, occur annually in the United States, among which approximately10% do not heal. In the orthopedic procedures conducted, about onemillion performed annually require allograft or autograft. One solutionto enhancement of bone healing is through tissue engineering, in whichcells, such as osteoblast, fibroblast, chondroblasts, are treated withbioactive signaling molecules, e.g., insulin or insulin mimetics orScaffolds such as β-TCP (tricalcium phosphate) and collagen under anappropriate environment. Current methods of treatment of bone fracturesinclude (a) electro-stimulation devices (such as PEMF, Exogen) and (b)biologics, such as bone morphogenic proteins (BMPs), e.g., rhBMP-2/ACS(INFUSE® Bone Graft). The latter has been approved by FDA as anautograft replacement in spine fusion (ALIF) with specific interbodycages (2002), as an adjuvant for repair of tibia fractures with IM nail(2004), and for craniofacial maxillary surgery (2006), but this methodis expensive, costing about $5,000 per application. (Lieberman, J. R.,et al., J. Bone Joint Surg. Am., 2002, 84: 1032-1044; Trippel, S. B., etal., J. Bone Joint Surg. Am., 1996, 78: 1272-86.)

Fracture healing is a complex process that involves the sequentialrecruitment of cells and the specific temporal expression of factorsessential for bone repair. The fracture healing process begins with theinitial formation of a blood clot at the fracture site. Platelets andinflammatory cells within the clot release several factors that areimportant for chemotaxis, proliferation, angiogenesis anddifferentiation of mesenchymal cells into osteoblasts or chondroblasts.

The fracture healing process subsequent to the initial hematomaformation can be classified as primary or secondary fracture healing.Primary fracture healing occurs in the presence of rigid internalfixation with little to no interfragmentary strain resulting in directbone formation across the fracture gap. Secondary fracture healingoccurs in response to interfragmentary strain due to an absence offixation or non-rigid fixation resulting in bone formation throughintramembranous and endochondral ossification characterized by responsesfrom the periosteum and external soft tissue.

Intramembranous bone formation originates in the periosteum. Osteoblastslocated within this area produce bone matrix and synthesize growthfactors, which recruit additional cells to the site. Soon after theinitiation of intramembranous ossification, the granulation tissuedirectly adjacent to the fracture site is replaced by cartilage leadingto endochondral bone formation. The cartilage temporarily bridging thefracture gap is produced by differentiation of mesenchymal cells intochondrocytes. The cartilaginous callus begins with proliferativechondrocytes and eventually becomes dominated by hypertrophicchondrocytes. Hypertrophic chondrocytes initiate angiogenesis and theresulting vasculature provides a conduit for the recruitment ofosteoblastic progenitors as well as chondroclasts and osteoclasts toresorb the calcified tissue. The osteoblastic progenitors differentiateinto osteoblasts and produce woven bone, thereby forming a unitedfracture. The final stages of fracture healing are characterized byremodeling of woven bone to form a structure, which resembles theoriginal tissue and has the mechanical integrity of unfractured bone.

However, the processes of bone metabolism are vastly different from bonerepair. Bone metabolism is the interplay between bone formation and boneresorption. Bone repair, as described previously, is a complex processthat involves the sequential recruitment and the differentiation ofmesenchymal cells towards the appropriate osteoblastic/chondrogeniclineage to repair the fracture/defect site.

SUMMARY OF THE INVENTION

The present invention provides unique strategies for bone healingthrough local administration of vanadium compounds as anabolic agents.

In one aspect the present invention provides a method of treating a bonecondition in a patient in need thereof, comprising locally administeringto the patient a therapeutically effective amount of an insulin-mimeticvanadium compound.

In another aspect the present invention provides a method of treating abone condition in a patient in need thereof, comprising locallyadministering to the patient a therapeutically effective amount of acomposition comprising an insulin-mimetic vanadium compound.

In another aspect the present invention provides use of aninsulin-mimetic vanadium compound for manufacture of a medicament foraccelerating bone healing in a patient in need thereof characterized bylocal administration of said medicament.

Preferably, the patient in need of bone healing is afflicted with a bonecondition selected from bone fracture, bone trauma, arthrodesis, and abone deficit condition associated with post-traumatic bone surgery,post-prosthetic joint surgery, post-plastic bone surgery, post-dentalsurgery, bone chemotherapy treatment, congenital bone loss, posttraumatic bone loss, post surgical bone loss, post infectious bone loss,allograft incorporation or bone radiotherapy treatment. More preferably,the bone condition is a bone fracture.

Preferably, the vanadium compound is an insulin mimetic organovanadiumcompound, such as vanadium compounds used for vanadium-based drugsvanadyl acetylacetonate and bis(ethylmaltolato)oxovanadium (IV), whichare insulin mimetic and have not been described for bone healing andfracture repair. Other preferred vanadium compounds also include, butare not limited to, vanadyl sulfate (VS), vanadyl 3-ethylacetylacetonate(VET), and vanadyl (IV) ascorbate complexes.

A more preferred vanadium compound for the present invention is vanadylacetylacetonate (VAC), an organovanadium compound that has demonstratedinsulin-mimetic effects in type 1 and type 2 diabetic animals and humanstudies, e.g., preventing some of the associated complications ofdiabetes in animal studies. Additional pharmacological activities of VACthat have been studied include inhibition of gluconeogenesis, a decreasein glutamate dehydrogenase activity, and antilipolysis.

An additional aspect of the present invention provides a drug deliverydevice or kit, which includes an insulin-mimetic vanadium compound and apharmaceutically acceptable carrier, wherein the device or kit isadapted for localized administration of the vanadium compound to apatient in need thereof. Localized delivery of vanadium-based insulinmimetics significantly enhanced biomechanical properties in 4 weeks, andthe outcome is dosage dependent, with lower dosages giving superiorresults.

Another additional aspect of the present invention includes localizedadministration of a vanadium compound or a composition thereof inconjunction with treating the patient with at least one additionalprocedure selected from bone autograft, bone allograft, autologous stemcell treatment, allogeneic stem cell treatment, chemical stimulation,electrical stimulation, internal fixation, and external fixation.

The present invention thus provides a unique method for promoting bonehealing in a patient, preferably mammalian animal and more preferably ahuman, either diabetic or non-diabetic. Development of a vanadiumtherapy of the present invention would obviate the need for developingspecialized methods to deliver growth factors and thereby reduce costsassociated with therapy, eliminate specialized storage, and enhance easeof use. These and other aspects of the present invention will be betterappreciated by reference to the following drawings and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows post-operative X-rays: Representative x-rays takenimmediately post-operative where (A) Einhorn model and (B) model areused in this work. (Note in (B) the Kirschner wire is going through thetrochanter, which helps to stabilize the fracture site and prevent themigration of the Kirschner wire).

FIG. 2 shows Mechanical Testing Setup: (A) intact femur before embeddedin ¾ inch square nut with Field's Metal, (B) intact femur embedded inhex nut and mounted in the mechanical testing apparatus, (C) intactfemur mounted in the mechanical testing apparatus after torsionaltesting, (D) intact femur after torsional testing, (E) fractured femurafter torsional testing showing spiral fracture indicative of healing,and (F) fractured femur after torsional testing showing non-spiralfracture indicative of non-union.

FIG. 3 shows x-ray photographs of femurs of saline vs. VAC after 4weeks.

FIG. 4 shows post-surgery bones where VN2 and VN3 represent two sets ofVAC-treated femurs harvested 4 weeks post-surgery (Left: FracturedFemur; Right: Intact Femur).

FIG. 5 shows spiral fractures after testing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on a hypothesis that vanadium can be usedto accelerate bone regeneration by stimulating insulin pathway signalingat a fracture site. In exploiting the biological impact of vanadium asan insulin-mimetic agent on bone, the present inventors found that theseagents play a critical role in bone healing. The present invention thususes an insulin-mimetic vanadium-based compound to treat various boneconditions, in particular, bone fractures.

In one aspect the present invention provides a method of treating a bonecondition in a patient in need thereof, comprising locally administeringto the patient a therapeutically effective amount of an insulin-mimeticvanadium compound.

In one embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein theinsulin-mimetic vanadium compound is an organovanadium compound selectedfrom the group consisting of vanadyl acetylacetonate (VAC), vanadylsulfate (VS), vanadyl 3-ethylacetylacetonate (VET), andbis(maltolato)oxovanadium (BMOV).

In a preferred embodiment of this aspect, the present invention providesa method of treating a bone condition in a patient, wherein theinsulin-mimetic vanadium compound is vanadyl acetylacetonate (VAC).

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein theinsulin-mimetic vanadium compound is directly administered to the boneinjury site.

Preferably, the patient in need of bone healing is afflicted with a bonecondition selected from bone fracture, bone trauma, arthrodesis, and abone deficit condition associated with post-traumatic bone surgery,post-prosthetic joint surgery, post-plastic bone surgery, post-dentalsurgery, bone chemotherapy treatment, congenital bone loss, posttraumatic bone loss, post surgical bone loss, post infectious bone loss,allograft incorporation or bone radiotherapy treatment.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the bonecondition is selected from bone fractures, osseous defects, and delayedunions and non-unions.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the method isused in conjunction with an allograft/autograft or orthopedicbiocomposite.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein saidinsulin-mimetic vanadium compound is locally administered by a bonegraft biocomposite containing said insulin-mimetic vanadium compound.

In another embodiment, the present invention provides a method oftreating a bone condition in a patient, wherein the bone graftbiocomposite is selected from the group consisting of autografts,allografts, xenografts, alloplastic grafts, and synthetic grafts. In apreferred embodiment, the bone graft is selected from the groupconsisting of autografts and allografts.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the methodcomprises co-administering a cytototoxic agent, cytokine or growthinhibitory agent with said vanadium compound.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the method isused in conjunction with an external bone growth stimulator, forexample, Exogen and Pulsed Electro Magnetic Field Therapy (PEMF).

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the methodco-administering a bioactive bone agent with said vanadium compound.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the method isused in conjunction with administration of a bioactive bone agentselected from the group consisting of peptide growth factors,anti-inflammatory factors, pro-inflammatory factors, inhibitors ofapoptosis, MMP inhibitors, and bone catabolic antagonists.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the method isused in conjunction with administration of a peptide growth factorselected from the group consisting of IGF (1,2), PDGF (AA, AB, BB),BMPs, FGF (1-20), TGF-beta (1-3), aFGF, bFGF, EGF, VEGF, parathyroidhormone (PTH), and parathyroid hormone-related protein (PTHrP).

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the method isused in conjunction with administration of an anti-inflammatory factorselected from the group consisting of anti-TNFa, soluble TNF receptors,ILlra, soluble IL1 receptors, IL4, IL-10, and IL-13.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the method isused in conjunction with administration of a bone catabolic antagonistselected from the group consisting of bisphosphonates, osteoprotegerin,and statins.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the patient isa mammalian animal.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the patient isa human.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein the patient isa non-diabetic human.

In another aspect, the present invention provides a method of treating abone condition in a patient, comprising locally administering to thepatient a therapeutically effective amount of a composition comprisingan insulin-mimetic vanadium compound.

In one embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein theinsulin-mimetic vanadium compound is an organovanadium compound.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein thecomposition further comprises at least one biocompatible carrier.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein thecomposition further comprises at least one biocompatible carrierselected from the group consisting of poly-lactic acid, poly-glycolicacid, and copolymers of poly-lactic acid and poly-glycolic acid.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein thecomposition further comprises at least one biocompatible carrierselected from the group consisting of biodegradable fatty acids andmetal salts of fatty acids.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein thecomposition further comprises at least one biocompatible carrierselected from the group consisting of palmitic acid, stearic acid, oleicacid, myristic acid, and metal salts thereof.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, wherein thecomposition further comprises at least one biocompatible carrierselected from the group consisting of porous or non-porous calciumphosphates, porous or non-porous hydroxyapatites, porous or non-poroustricalcium phosphates, porous or non-porous tetracalcium phosphates, andporous or non-porous calcium sulfates, or a combination thereof.

In another embodiment of this aspect, the present invention provides amethod of treating a bone condition in a patient, comprising locallyadministering to the patient a therapeutically effective amount of acomposition comprising a vanadium compound and further comprisingtreating said patient with at least one procedure selected from thegroup consisting of bone autograft, bone allograft, autologous stem celltreatment, allogeneic stem cell treatment, chemical stimulation,electrical stimulation, internal fixation, and external fixation.

In another aspect the present invention provides use of aninsulin-mimetic vanadium compound for manufacture of a medicament foraccelerating bone healing in a patient in need thereof characterized bylocal administration of said medicament.

Preferably, the patient in need of bone healing is afflicted with a bonecondition selected from bone fracture, bone trauma, arthrodesis, and abone deficit condition associated with post-traumatic bone surgery,post-prosthetic joint surgery, post-plastic bone surgery, post-dentalsurgery, bone chemotherapy treatment, congenital bone loss, posttraumatic bone loss, post surgical bone loss, post infectious bone loss,allograft incorporation or bone radiotherapy treatment.

In one embodiment of this aspect the present invention provides use of avanadium compound for manufacture of a medicament for accelerating bonehealing in a patient in need thereof characterized by localadministration of said medicament, wherein the insulin-mimetic vanadiumcompound is an inorganic vanadium compound.

In another embodiment of this aspect the present invention provides useof a vanadium compound for manufacture of a medicament for acceleratingbone healing in a patient in need thereof characterized by localadministration of said medicament, wherein the insulin-mimetic vanadiumcompound is an organovanadium compound.

In one preferred embodiment of this aspect the present inventionprovides use of a vanadium compound for manufacture of a medicament foraccelerating bone healing in a patient in need thereof characterized bylocal administration of said medicament, wherein the insulin-mimeticvanadium compound is an organovanadium compound selected from the groupconsisting of vanadyl acetylacetonate (VAC), vanadyl sulfate (VS),vanadyl 3-ethylacetylacetonate (VET), and bis(maltolato)oxovanadium(BMOV).

In another aspect the present invention provides a bone injury treatmentkit comprising a therapeutically effective amount of an insulin-mimeticvanadium compound formulated for localized administration of saidvanadium compound to a patient in need thereof.

In one embodiment of this aspect the present invention provides a boneinjury treatment kit, wherein the insulin-mimetic vanadium compound isan organovanadium compound.

In another embodiment of this aspect the present invention provides abone injury treatment kit, wherein the insulin-mimetic vanadium compoundis selected from the group consisting of vanadyl acetylacetonate (VAC),vanadyl sulfate (VS), vanadyl 3-ethylacetylacetonate (VET), andbis(maltolato)oxovanadium (BMOV).

In another embodiment of this aspect the present invention provides abone injury treatment kit, wherein the insulin-mimetic vanadium compoundis vanadyl acetylacetonate (VAC).

In another embodiment of this aspect the present invention provides abone injury treatment kit, wherein the patient is a mammalian animal.

In another embodiment of this aspect the present invention provides abone injury treatment kit, wherein the patient is a human.

In another embodiment of this aspect the present invention provides abone injury treatment kit, wherein the patient is a non-diabetic human.

In another embodiment of this aspect the present invention provides abone injury treatment kit, wherein the patient is an animal selectedfrom horses, dogs, and cats.

In another embodiment of this aspect the present invention provides abone injury treatment kit, further comprising a bone graft biocomposite.

In another embodiment of this aspect the present invention provides abone injury treatment kit, further comprising a bioactive agent fortreating injured bone tissue.

Preferred sites of interest in the patient include sites in need of bonehealing and areas adjacent and/or contiguous to these sites. Optionally,the treatment method of the present invention is combined with at leastone procedure selected from bone autograft, bone allograft, autologousstem cell treatment, allogeneic stem cell treatment, chemicalstimulation, electrical stimulation, internal fixation, and externalfixation.

The vanadium compounds of the invention can be any organic vanadiumcompound known to increase safety. improve absorption, and reduceundesirable. side effects associated with therapeutic vanadium, forexample, vanadyl sulfate (VS), vanadyl 3-ethylacetylacetonate (VET),bis(maltolato)oxovanadium (BMOV), and vanadyl acetylacetonate (VAC).Advantages of an insulin mimetic include, but are not limited to: (a)development of a small molecule insulin mimetic can be of greatsignificance to bone fracture patients; (b) insulin composite whichrequires a carrier may be difficult to meet FDA requirements as a dualagent product; and (c) vanadium salts may have longer half life andavoid the storage issues commonly seen with proteins.

Vanadium, which exists in +4 (vanadyl) and +5 (vanadate) compounds inthe biological body, have demonstrated poor absorption rates within thegastrointestinal (GI) tract and GI side-effects, such as diarrhea andvomiting. As a result, additional organic vanadium compounds, i.e.,vanadyl sulfate (VS), vanadyl 3-ethylacetylacetonate (VET),bis(maltolato)oxovanadium (BMOV), and VAC, have been synthesized inorder to improve absorption and safety (Poucheret, P., et al., Mol. CellBiochem., 1998, 188(1-2): 73-80). VAC with an organic ligand has beenproven to be more effective in its anti-diabetic function compared withother vanadium compounds, including BMOV, VS, and VET (Reul, B. A., etal., Br. J. Pharmacol. 1999, 126(2):467-477). Unfortunately, in thesestudies the concentration and doses of the vanadium complexes were notclosely regulated to the exact dosage required to obtain insulin-mimeticeffects remain unclear. The doses delivered in the present study areclosely regulated and evaluated at various time points. Our labdemonstrated full animal survivorship at 4 weeks with no indication ofsickness, diarrhea, or vomiting after a single local dose administrationsuggest the safety of this treatment.

Exemplary healing mechanisms include, but are not limited to: (a)retaining mineralized components in bone, (b) inhibiting release ofmineralized components from bone, (c) stimulating osteoblast activity,(d) reducing osteoclast activity, or (e) stimulating bone remodeling.

The term “therapeutically effective amount,” as used herein, means anamount at which the administration of an agent is physiologicallysignificant. The administration of an agent is physiologicallysignificant if its presence results in a detectable change in the bonehealing process of the patient.

The term “bone injury,” “injured bone,” or the like, as used herein,refers to a bone condition selected from the group consisting of bonefracture, bone trauma, arthrodesis, and a bone deficit conditionassociated with post-traumatic bone surgery, post-prosthetic jointsurgery, post-plastic bone surgery, post-dental surgery, bonechemotherapy treatment, congenital bone loss, post traumatic bone loss,post surgical bone loss, post infectious bone loss, allograftincorporation or bone radiotherapy treatment.

It will be appreciated that actual preferred amounts of a pharmaceuticalcomposition used in a given therapy will vary depending upon theparticular form being utilized, the particular compositions formulated,the mode of application, and the particular site of -administration, andother such factors that are recognized by those skilled in the artincluding the attendant physician or veterinarian. Optimaladministration rates for a given protocol of administration can bereadily determined by those skilled in the art using conventional dosagedetermination tests.

Dosages of a vanadium compound employable with the present invention mayvary depending on the particular use envisioned. The determination ofthe appropriate dosage or route of administration is well within theskill of an ordinary physician.

For example, when in vivo administration of a vanadium compound isemployed, normal dosage amounts may vary from about 10 ng/kg up to about100 mg/kg of mammal body weight or more per day, preferably about 1g/kg/day to 10 mg/kg/day, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, U.S. Pat. No. 4,657,760; U.S. Pat. No.5,206,344 or U.S. Pat. No. 5,225,212. It is anticipated that differentformulations will be effective for different treatments and differentdisorders, and that administration intended to treat a specific bonesite or condition, may necessitate delivery in a manner different fromthat for another site or condition.

The formulations used herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the formulation maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are present in combinations and amounts that are effective forthe intended purpose.

Therapeutic formulations of vanadium compounds in the vanadium deliverysystems employable in the methods of the present invention are preparedfor storage by mixing the vanadium compound having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)). Such therapeutic formulations can be in the form oflyophilized formulations or aqueous solutions. Acceptable biocompatiblecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and may include buffers, forexample, phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (e.g.,octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, for example, methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); lowmolecular weight (less than about 10 residues) polypeptides; proteins,for example, serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers, for example, polyvinylpyrrolidone; amino acids, for example,glycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, dextrins, or hyaluronan; chelating agents, forexample, EDTA; sugars, for example, sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions, for example, sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactants,for example, TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In order for the formulations to be used for in vivo administration,they must be sterile. The formulation may be readily rendered sterile byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The therapeutic formulations hereinpreferably are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

The formulations used herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the formulation maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are present in combinations and amounts that are effective forthe intended purpose.

Optionally, the vanadium delivery system includes a bioactive bone agentin addition to vanadium insulin-mimetic agent. Preferably, the bioactivebone agent is selected from peptide growth factors (e.g., IGF (1, 2),PDGF (AA, AB, BB), BMPs, FGF (1-20), TGF-beta (1-3), aFGF, bFGF, EGF,and VEGF), anti-inflammatory factors (e.g., anti-TNFα, soluble TNFreceptors, ILlra, soluble IL1 receptors, IL4, IL-10, and IL-13),pro-inflammatory factors, inhibitors of apoptosis, MMP inhibitors andbone catabolic antagonists (e.g., bisphosphonates and osteoprotegerin).

The vanadium may also be entrapped in microcapsules prepared, forexample by coacervation techniques or by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively. Such preparationscan be administered in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th Edition (or newer), Osol A.Ed. (1980).

Optionally, vanadium in the vanadium delivery systems includes a porouscalcium phosphate, non-porous calcium phosphate, hydroxyapatite,tricalcium phosphate, tetracalcium phosphate, calcium sulfate, calciumminerals obtained from natural bone, inorganic bone, organic bone, or acombination thereof.

Where sustained-release or extended-release administration of vanadiumin the vanadium delivery systems is desired, microencapsulation iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon-α, -β, -γ (rhIFN-α, -β, -γ), interleukin-2, and MNrgp120. (Johnson et al., Nat. Med., 1996, 2:795-799; Yasuda, Biomed.Ther., 1993, 27:1221-1223; Hora et al., Bio/Technology, 1990, 8:755-758;Cleland, “Design and Production of Single Immunization Vaccines UsingPolylactide Polyglycolide Microsphere Systems” in Vaccine Design: TheSubunit and Adjuvant Approach, Powell and Newman, eds., Plenum Press:New York, 1995, pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399 andU.S. Pat. No. 5,654,010).

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thevanadium in the vanadium delivery systems, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include one or more polyanhydrides (e.g.,U.S. Pat. Nos. 4,891,225 and 4,767,628), polyesters, for example,polyglycolides, polylactides and polylactide-co-glycolides (e.g., U.S.Pat. Nos. 3,773,919; 4,767,628; and 4,530,840; Kulkarni et al., Arch.Surg., 1996, 93:839), polyamino acids, for example, polylysine, polymersand copolymers of polyethylene oxide, polyethylene oxide acrylates,polyacrylates, ethylene-vinyl acetates, polyamides, polyurethanes,polyorthoesters, polyacetylnitriles, polyphosphazenes, and polyesterhydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), cellulose, acyl substituted cellulose acetates,non-degradable polyurethanes, polystyrenes, polyvinyl chloride,polyvinyl fluoride, poly(vinylimidazole), chlorosulphonated polyolefins,polyethylene oxide, copolymers of L-glutamic acid and.gamma.-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers, for example, the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release for over 100 days, certain hydrogels releaseproteins for shorter time periods. Additional non-biodegradable polymerswhich may be employed are polyethylene, polyvinyl pyrrolidone, ethylenevinylacetate, polyethylene glycol, cellulose acetate butyrate andcellulose acetate propionate.

Alternatively, sustained-release formulations may be composed ofdegradable biological materials, for example, bioerodible fatty acids(e.g., palmitic acid, stearic acid, oleic acid, and the like).Biodegradable polymers are attractive drug formulations because of theirbiocompatibility, high responsibility for specific degradation, and easeof incorporating the active drug into the biological matrix. Forexample, hyaluronic acid (HA) may be crosslinked and used as a swellablepolymeric delivery vehicle for biological materials. (U.S. Pat. No.4,957,744; Valle et al., Polym. Mater. Sci. Eng., 1991, 62:731-735). HApolymer grafted with polyethylene glycol has also been prepared as animproved delivery matrix which reduced both undesired drug leakage andthe denaturing associated with long term storage at physiologicalconditions. (Kazuteru, M., J. Controlled Release, 1999, 59:77-86).Additional biodegradable polymers which may be used arepoly(caprolactone), polyanhydrides, polyamino acids, polyorthoesters,polycyanoacrylates, poly(phosphazines), poly(phosphodiesters),polyesteramides, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, degradable and nontoxicpolyurethanes, polyhydroxylbutyrates, polyhydroxyvalerates, polyalkyleneoxalates, polyalkylene succinates, poly(malic acid), chitin, andchitosan.

Alternatively, biodegradable hydrogels may be used as controlled-releasematerials for the vanadium compounds in the vanadium delivery systems.Through the appropriate choice of macromers, membranes can be producedwith a range of permeability, pore sizes and degradation rates suitablefor different types of vanadium compounds in the vanadium deliverysystems.

Alternatively, sustained-release delivery systems for vanadium in thevanadium delivery systems can be composed of dispersions. Dispersionsmay further be classified as either suspensions or emulsions. In thecontext of delivery vehicles for a vanadium compound, suspensions are amixture of very small solid particles which are dispersed (more or lessuniformly) in a liquid medium. The solid particles of a suspension canrange in size from a few nanometers to hundreds of microns, and includemicrospheres, microcapsules and nanospheres. Emulsions, on the otherhand, are a mixture of two or more immiscible liquids held in suspensionby small quantities of emulsifiers. Emulsifiers form an interfacial filmbetween the immiscible liquids and are also known as surfactants ordetergents. Emulsion formulations can be both oil in water (o/w) whereinwater is in a continuous phase while the oil or fat is dispersed, aswell as water in oil (w/o), wherein the oil is in a continuous phasewhile the water is dispersed. One example of a suitablesustained-release formulation is disclosed in WO 97/25563. Additionally,emulsions for use with a vanadium compound in the present inventioninclude multiple emulsions, microemulsions, microdroplets and liposomes.Microdroplets are unilamellar phospholipid vesicles that consist of aspherical lipid layer with an oil phase inside. (E.g., U.S. Pat. No.4,622,219 and U.S. Pat. No. 4,725,442). Liposomes are phospholipidvesicles prepared by mixing water-insoluble polar lipids with an aqueoussolution.

Alternatively, the sustained-release formulations of vanadium in thevanadium delivery systems may be developed using poly-lactic-co-glycolicacid (PLGA), a polymer exhibiting a strong degree of biocompatibilityand a wide range of biodegradable properties. The degradation productsof PLGA, lactic and glycolic acids, are cleared quickly from the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition. Forfurther information see Lewis, “Controlled Release of Bioactive Agentsfrom Lactide/Glycolide polymer,” in Biodegradable Polymers as DrugDelivery Systems, M. Chasin and R. Langeer, eds. (Marcel Dekker: NewYork, 1990), pp. 1-41.

The route of administration of “local vanadium” via “insulin mimeticdelivery system” is in accordance with known methods, e.g. viaimmediate-release, controlled-release, sustained-release, andextended-release means. Preferred modes of administration for theinsulin-mimetic delivery system include injection directly intoafflicted bone site and areas adjacent and/or contiguous to these siteor surgical implantation of insulin-mimetic delivery system directlyinto afflicted bone sites and area adjacent and/or contiguous to thesesite. This type of system may allow temporal control of release as wellas location of release as stated above.

As an illustrated example, Vanadium may be continuously administeredlocally to a site via a delivery pump. In one embodiment, the pump isworn externally (in a pocket or on the belt) and attached to the bodywith a long, thin, and flexible plastic tubing that has a needle or softcannula (thin plastic tube), and the cannula or needle is inserted andthen left in place beneath the skin. The needle or cannula and tubingcan be changed, for example, every 48 to 72 hours. The pump would storethe vanadium in a cartridge and release it based on the optimal deliveryrate. Optionally, the pump is programmed to give a small dose of a drugcontinuously through the day and night, which in certain circumstancesmay be preferred.

The present invention may prove effective at treating both non-unionsand delayed unions. Because up to 10% of the 6.2 million fracturessustained annually proceed to delayed union and non-union (Praemer, A.and Rice D., Am. Acad. Orthop. Surg., 1992, 85-124), the presentinvention may find wide applications.

In one preferred embodiment, the present invention can be used fortreating military injuries. In the recent United States conflicts,significant improvements in personal body armor have led to fewercasualties. While this advancement in personal protection has reducedthe number of mortalities, the morbidity of war, specifically adramatically larger portion of battle-related injuries, has occurred inthe extremities. Depending upon the level of energy, extremity fracturemay range from simple closed fracture to large segmental defects with asignificant bone and soft tissue loss evident. Battle-related fractureshave very high complication rates (47% in one study) with delayed unionand non-union in 31% of all the fractures followed (Pukljak D., J.Trauma., 1997, 43(2):275-282). Many of these fractures occur in theextremities. Bullet wounds are often severe due to the large amount ofkinetic energy expended on the bone surface.

Current simple and comminuted fracture treatment relies upon restoringthe bone's anatomy and stabilizing the fractured bone until the body isable to heal the fracture with newly produced bone. Adjuncts to thisbasic procedure such as a method to significantly enhance boneregeneration and while maintaining appropriate blood flow and preventinginfection have the potential to revolutionize this field. Osseous agentssuch as vanadium may enhance fracture callus strength by exploiting thehealing responsiveness of insulin pathways. Localized therapy of thisnon-protein agent has a minimal possibility of infection or systemicconsequences associated with systemic treatments.

The high complication rate of severe military injuries with delayedunion and non-unions parallels the observations seen in the civilianpopulation, who have risk factors for impaired bone healing. Riskfactors include smoking, old age, steroid use, certain pharmaceuticals(i.e. anti-cancer drugs) and diabetes mellitus (DM). Treatment methodsuitable for the impaired osseous healing associated with high-riskpopulations can also be used to accelerate fracture healing in thenormal, young, healthy soldiers.

In another preferred embodiment, the present invention may find wideapplication in sports medicines to treat a variety of fractures,including fatigue fractures and acute sports-related fractures. Acutefractures occurring during athletics result from overloading bone (boottop tibial fractures in skiing) or from ligament to tendon avulsion(tibial tubercle avulsion during long jumping). High school footballinjuries alone account for over 38,000 annual fractures (DeCoster, T.A., et al., Iowa Orthop. J., 1994, 14:81-84). Sports fractures include,but are not limited to, tibial (49%), femoral (7%), and tarsal (25%)fractures which may differ depending on the individual and cause ofinjury (DeCoster, T. A., et al., Iowa Orthop. J., 1994, 14:81-84). Thepresent work examined a mid-diaphyseal fracture pattern, but it islikely that other fracture patterns would heal in the same fashion.

In yet another embodiment, the present invention may find wideapplication in veterinary medicines to treat a variety of fractures in amammalian animal, including but not limited to, horses, dogs, cats, orany other domestic or wild mammalian animals. A particular usefulapplication may be found, for example, in treating an injured racehorse.

The following non-limiting examples illustrate certain aspects of theinvention.

EXAMPLES Materials and Methods The BB Wistar Rat Model Animal Source andOrigin

Diabetic Resistance (DR) BB Wistar rats used in the study were obtainedfrom a breeding colony at UMDNJ-New Jersey Medical School (NJMS). Therats were housed under controlled environmental conditions and fed adlibitum.

Diabetic Resistant BB Wistar Rats

A total of 86 DR BB Wistar rats were utilized in the study. Twentysamples were excluded from the study, fifteen due to inappropriatelyfractured condyles and five due to movement in potting during testing.The remaining 66 animals were used for mechanical testing, distributedamongst the control saline (n=6), calcium sulfate buffer (n=9), 0.25mg/kg VAC (n=6), 0.5 mg/kg VAC (n=6), 1.5 mg/kg VAC (n=15), 3 mg/kg VAC(n=13), 0.25 mg/kg VAC with calcium sulfate carrier (n=6), and 1.5 mg/kgVAC with calcium sulfate carrier (n=5) groups. At sacrifice, 2 ml ofwhole blood was collected and glucose and percent glycosylatedhemoglobin (% HbA1c) levels were determined using a glycosylatedhemoglobin kit (Bayer HealthCare, Sunnyvale, Calif.). Glycosylatedhemoglobin is a time-averaged measure of blood glucose control and canbe twice as high in patients with poor blood glucose control whencompared to normal patients.

Closed Femoral Fracture Model

Surgery was performed in DR animals between ages 80 and 120 days, usinga closed mid-diaphyseal fracture model, on the right femur as describedpreviously.

General anesthesia was administrated by intraperitoneal (IP) injectionof ketamine (60 mg/kg) and xylazine (8 mg/Kg). The right leg of each ratwas shaved and the incision site was cleansed with Betadine and 70%alcohol. An approximately 1 cm medial, parapatellar skin incision wasmade over the patella. The patella was dislocated laterally and theinterchondylar notch of the distal femur was exposed. An entry hole wasmade with an 18 gauge needle and the femur was reamed with the 18 gaugeneedle. A Kirschner wire (316LVM stainless steel, 0.04 inch diameter,Small Parts, Inc., Miami Lakes, Fla.) was inserted the length of themedullary canal, and drilled through the trochanter of the femur. TheKirschner wire was cut flush with the femoral condyles. Afterirrigation, the wound was closed with 4-0 vicryl resorbable suture. Aclosed midshaft fracture was then created unilaterally with the use of athree-point bending fracture machine. X-rays were taken to determinewhether the fracture is of acceptable configuration. An appropriatefracture is an approximately mid-diaphyseal, low energy, transversefracture (FIG. 1). The rats were allowed to ambulate freely immediatelypost-fracture. This closed fracture model is commonly used to evaluatethe efficacy of osseous wound healing devices and drugs.

Experimental Treatments Local Vanadium Delivery

A 0.1 ml solution of Vanadium (Vanadyl acetylacetonate (VAC), SigmaAldrich, St. Louis, Mo.) was injected into the intramedullary canalprior to fracture mixed with sterile water at concentrations of 0.725 mgVAC/ml water (extra low dose), 1.45 mg VAC/ml water (lowered low dose),4.35 mg VAC/ml water (low dose), and 8.7 mg VAC/ml water (high dose).The extra low dose correlated to a dose of 0.25 mg/ml, the lowered lowdose correlated to 0.5 mg/kg, the low dose correlated to 1.5 mg/kg andhigh dose correlated to 3.0 mg/kg. The analysis of low dose and highdose VAC in combination with calcium sulfate (CaSO₄) hemihydrate (J. T.Baker) will be described below.

Local Vanadium Treatment using CaSO₄ as a Carrier

Two grams of CaSO₄ were placed in glass vials. The vials were placed ina mechanical convection oven and sterilized at 196° C. for 6 hours.

To prepare the CaSO₄-VAC mixture, 0.8 g of CaSO₄ was mixed with 400 μlof saline or the respective VAC solutions for one minute at roomtemperature. The mixture was packed into the barrel of a 1 cc sterilesyringe and pushed down to the open orifice of the syringe barrel byinsertion of the syringe plunger. After attaching an 18-gauge sterileneedle to the syringe barrel, 0.1 ml volume of the mixture was directlyinjected into the rat femoral canal (non-diabetic BB Wistar rat) priorto Kirschner wire insertion and fracture.

Four experimental groups were used:(1) Saline Control (0.9% NaCl): 0.1 ml of saline was injected into theintramedullary canal of the right femur prior to the Kirschner wireinsertion and fracture.(2) Calcium Sulfate Buffer Control: 0.1 ml of the CaSO₄-Saline mixturewas injected into the intramedullary canal of the right femur prior toKirschner wire insertion and fracture.(3) 0.25 mg/kg Local VAC+Carrier: 0.1 ml of the CaSO₄-VAC (Vanadylacetylacetonate (VAC), Sigma Aldrich, St. Louis, Mo.) mixture wasinjected into the intramedullary canal of the right femur prior toKirschner wire insertion and fracture.(4) 1.25 mg/kg Local VAC+Carrier: 0.1 ml of the CaSO₄-VAC (Vanadylacetylacetonate (VAC), Sigma Aldrich, St. Louis, Mo.) mixture wasinjected into the intramedullary canal of the right femur prior toKirschner wire insertion and fracture.

Glycosylated Hemoglobin (HbA1c)

Glycosylated hemoglobin (HbA1c) is formed by the nonenzymatic reactionbetween glucose and the globin chains of hemoglobin. Specifically,glucose forms a non-covalent bond with an amine group found in thehemoglobin molecule. This reaction occurs continuously throughout thelife-cycle of the red blood cell and, therefore, becomes a measure ofaverage blood glucose levels during the life of the cell. HbA1c has beenaccepted as a time-averaged measure of blood glucose control and can betwice as high in patients with poor blood glucose control when comparedto normal patients.

Blood was collected from rats at sacrifice by cardiac puncture.Approximately 2 ml of whole blood was collected and the percentglycosylated hemoglobin (% HbA1c) was determined using a glycosylatedhemoglobin kit (Bayer HealthCare, Sunnyvale, Calif.) as per themanufacturer's instructions.

Mechanical Testing

X-rays were taken at 4 weeks to determine the extent of healing of thefractures (FIG. 3).

Fractured and contralateral femora were resected at 4 and 5 weekspost-fracture. Femora were cleaned of soft tissue and the intramedullaryrod was removed. Samples were wrapped in saline (0.9% NaCl) soaked gauzeand stored at −20° C. Prior to testing, all femora were removed from thefreezer and allowed to thaw to room temperature for three to four hours.The proximal and distal ends of the fractured and contralateral femorawere embedded in ³A inch square nuts with Field's Metal, leaving anapproximate gauge length of 12 mm (FIG. 2). After measuring callus andfemur dimensions, torsional testing was conducted using aservohydraulics machine (MTS Systems Corp., Eden Prairie, Minn.) with a20 Nm reaction torque cell (Interface, Scottsdale, Ariz.) and tested tofailure at a rate of 2.0 deg/sec. The maximum torque to failure andangle to failure were determined from the force to angular displacementdata.

Peak torque to failure (T_(max)), torsional rigidity (TR), effectiveshear modulus (SM), and effective torsional shear stress (SS) werecalculated through standard equations. T_(max) and TR are consideredextrinsic properties while SM and SS are considered intrinsicproperties. T_(max) was defined as the point where an increase inangular displacement failed to produce any further increase in torque.TR is a function of the torque to failure, gauge length (distance of theexposed femur between the embedded proximal and distal end) and angulardisplacement. SS is a function of the torque to failure, maximum radiuswithin the mid-diaphyseal region and the polar moment of inertia. Thepolar moment of inertia was calculated by modeling the femur as a hollowellipse. Engesaeter et al. (Acta Orthop. Scand., 1978, 49(6):512-518)demonstrated that the calculated polar moment of inertia using thehollow ellipse model differed from the measured polar moment of inertiaby only 2 percent (Engesaeter et al., Acta Orthop. Scand., 1978,49(6):512-518).

In order to compare the biomechanical parameters between differentgroups, the data was normalized by dividing each fractured femur valueby its corresponding intact, contralateral femur value (FIG. 2).Normalization was used to minimize biological variability due todifferences in age and weight among rats.

In addition to the biomechanical parameters determined through torsionaltesting, the mode of failure can also provide substantial information.The mode of torsional failure as determined by gross inspection providedan indication as to the extent of healing (FIG. 4). A spiral failure inthe mid-diaphyseal region indicated a complete union while a transversefailure through the fracture site indicated a nonunion. A combinationspiral/transverse failure indicated a partial union (FIG. 5).

Data and Statistical Analysis

Analysis of variance (ANOVA) was performed followed by Holm-Sidakpost-hoc tests to determine differences (SigmaStat 3.0, SPSS Inc.,Chicago, Ill.). A P value less than 0.05 was considered statisticallysignificant.

Results General Health

In this biomechanical testing experiment, animals among treatment groupswere age matched. There was no statistical difference among treatmentgroups in percent weight gain from the time of surgery indicating thatvanadium injected locally into the intramedullary canal had no effect onmetabolism (Table 1). Blood glucose levels and age at surgery showed asignificant difference between the high dose and saline groups (Table 2)however, the clinical relevance of this observation is difficult toascertain since this range is within the normoglycemic value of Non-DMrats. These fluctuations may be a result of the small sample size andvariations based on diet.

All animals were grouped within the same age within 40 days (80-120days). The average difference in age between groups was less than 12days between the high dose and saline treated groups, and less thanthree days between low dose and control treated groups. Such a small agedifference within this phase is an unlikely factor to cause any majorvariances in healing rates.

TABLE 1 General Health of Non-DM BB Wistar Rats: Local Vanadium (VAC)Delivery without a Carrier (4 Week Mechanical Testing) Blood Glucose(mg/dl)* Age % Weight Pre-Surgery At Surgery gain Saline (n = 5)  81.7 ±4.3 99.0 ± 1.0^(b) 3.5 ± 2.3 (day 1) Low Dose  95.1 ± 6.1 96.3 ± 7.5 7.7± 6.5 (n = 6) High Dose 107.6 ± 19.1^(a) 88.2 ± 5.0 8.1 ± 7.2 (n = 5)The data represents average values ± standard deviation ^(a)representsvalues significantly greater than the saline group; p = 0.003^(b)represents values significantly greater than the high dose group; p= 0.008

Mechanical Testing Results

The effect of local vanadium therapy on healing of femur fractures innormal (non-diabetic) rats was measured by torsional mechanical testing.At 4 weeks post-fracture, rats treated with between 1.5 mg/kg and 3mg/kg VAC displayed improved mechanical properties of the fracturedfemora compared to the untreated saline group. The maximum torque tofailure (P<0.05), maximum torsional rigidity (P<0.05), effective shearstress (P<0.05), and effective shear modulus (P<0.05) were allsignificantly increased compared to the untreated group (Table 2).Maximum torque to failure and maximum torsional rigidity were alsosignificantly increased (P<0.05) for the 0.5 mg/kg VAC compared to theuntreated saline group. When the mechanical parameters of the fracturedfemora were normalized to the intact, contralateral femora, percenttorque to failure (P<0.05), percent maximum torsional rigidity (P<0.05HD), and percent effective shear modulus (P<0.05) were significantlygreater for several of the local vanadium treated groups when comparedto the saline group. (Table 2).

At 5 weeks post-fracture, rats treated with 1.5 mg/kg displayed improvedmechanical properties of the fractured femora compared to the untreatedsaline group and the 3 mg/kg VAC treated group. The maximum torque tofailure (P<0.05), maximum torsional rigidity (P<0.05) were allsignificantly increased compared to the untreated group. The 1.5 mg/kgVAC group also demonstrated a significantly higher maximum torque tofailure (P<0.05) compared to the 3 mg/kg VAC group (Table 3). When themechanical parameters of the fractured femora were normalized to theintact, contralateral femora, although several parameters approachedsignificance, none were determined to be significant (Table 3).

At 4 weeks post-fracture, rats treated with 0.25 mg/kg VAC and a calciumsulfate carrier displayed improved mechanical properties of thefractured femora compared to the untreated saline group and the calciumsulfate buffer group. The maximum torque to failure (P<0.05), andeffective shear modulus (P<0.05) were both significantly increasedcompared to the untreated group, while the effective shear stress wassignificantly increased (P<0.05) for the 0.25 mg/kg VAC w/CalciumSulfate group compared to the untreated saline, calcium sulfate buffer,and 1.5 mg/kg VAC w/Calcium Sulfate groups (Table 4). When themechanical parameters of the fractured femora were normalized to theintact, contralateral femora, percent torque to failure (P<0.05), andpercent effective shear modulus (P<0.05) were significantly greater forthe 0.25 mg/kg VAC w/Calcium Sulfate group compared to the untreatedsaline, and calcium sulfate buffer groups (Table 2). Percent maximumtorsional rigidity was significantly higher (P<0.05) for the 0.25 mg/kgVAC w/Calcium Sulfate group compared to the untreated saline group(Table 4).

To our knowledge, this is the first study to examine the effect of localvanadium treatment on fracture healing, quantified by mechanicaltesting. Our study demonstrated that local VAC treatment significantlyimproved the biomechanical parameters of fracture healing in normalanimals (Tables 2-4).

At four weeks the average percent torque to failure of the fracturedfemora for both 1.5 mg/kg and 3.0 mg/kg doses with no carrier weresignificantly greater, with 3.0 mg/kg dose 1.93 times greater (79.0% ofcontralateral vs. 27.0%), and 1.19 times greater (59.0% of contralateralvs. 27.0%) compared to the untreated saline group. Percent maximumtorsional rigidity values for both 1.5 mg/kg and 3.0 mg/kg doses weresignificantly greater, with 3.0 mg/kg dose 2.90 times greater (78.0% ofcontralateral vs. 20.0%), and 2.80 times greater (76.0% of contralateralvs. 20.0%) compared to the untreated saline group. Percent effectiveshear modulus values for both 1.5 mg/kg and 3.0 mg/kg doses weresignificantly greater, with high dose 4.75 times greater (23.0% ofcontralateral vs. 4.0%), and 4 times greater (20.0% of contralateral vs.4.0%) compared to the untreated saline group. Percent effective shearstress values for the 3.0 mg/kg dose was significantly greater, 2 timesgreater (30.0% of contralateral vs. 10.0%), compared to the untreatedsaline group.

At four weeks with the introduction of an industry standard carrier(Calcium Sulfate), the average percent torque to failure of thefractured femora for the 0.25 mg/kg dose were significantly greater thanboth the untreated saline, 2.15 times greater (85.0% of contralateralvs. 27.0%) and Calcium Sulfate Buffer, 1.30 times greater (85.0% ofcontralateral vs. 37.0%) groups. Percent maximum torsional rigidity forthe 0.25 mg/kg dose was significantly greater than the untreated saline,4 times greater (100.0% of contralateral vs. 20.0%). Percent effectiveshear modulus values for the 0.25 mg/kg dose were significantly greaterthan both the untreated saline, 5 times greater (24.0% of contralateralvs. 4.0%) and Calcium Sulfate Buffer, 2.43 times greater (24.0% ofcontralateral vs. 7.0%) groups.

These results exemplify the incredible potential of local VAC treatmentfor non-diabetic fracture healing. They show that lower doses of VACwith a carrier may optimize its delivery, and its reduce effective dose.Further we show a near dose dependent response to VAC delivery, withhigher doses leading to enhanced biomechanical parameters. At five weekspost fracture, it is clear that there is a limit to the effectiveness ofvanadium compounds the highest dose tested in terms of callus size andbone remodeling (Table 4). An earlier study examining the effect ofvanadium on mechanical strength of intact (non fractured) bone innon-diabetic and diabetic animals revealed that vanadium had no effecton bone homeostasis in non-diabetic animals (Facchini, D. M., et al.,Bone, 2006, 38(3):368-377). The fracture healing pathway is differentthan the bone homeostasis pathway. This is likely the primary reason forconflicting results presented in both models. Other possibilitiesinclude different dosages and delivery methods in each study.

TABLE 2 Four weeks post-fracture mechanical testing with Vanadium (VAC)in normal rats Maximum Maximum Torque to Torsional Rigidity EffectiveShear Effective Shear failure (Nmm) (Nmm²/rad) Modulus (MPa) Stress(MPa) Control (n = 6) 161 ± 48  9,889 ± 4,719 258 ± 108 17 ± 4 0.25mg/kg VAC (n = 6) 227 ± 64 28,218 ± 9,107 878 ± 416 25 ± 9  0.5 mg/kgVAC (n = 6) 362 ± 49 *^(, #) 45,877 ± 13,079 * 1,107 ± 441    32 ± 13 1.5 mg/kg VAC (n = 6) 329 ± 117 * 34,526 ± 16,851 * 2,454 ± 2,370 * 69± 59 * 3.0 mg/kg (n = 5) 409 ± 43 *^(, #) 41,007 ± 11,236 * 2,948 ±1,218 * 101 ± 18 *^(, #, &) Fractured femur values normalized to thecontralateral (intact) femur Percent maximum Percent torque to Percentmaximum Percent Effective Effective Shear failure torsional rigidityShear Modulus Stress Control (n = 6) 27 ± 18 20 ± 10  4 ± 2 10 ± 5 0.25mg/kg VAC (n = 6) 49 ± 14 67 ± 21 * 14 ± 4 10 ± 3  0.5 mg/kg VAC (n = 6)72 ± 19 * 103 ± 23 *^(, #) 16 ± 7  20 ± 11  1.5 mg/kg VAC (n = 6) 59 ±28 76 ± 28 * 23 ± 12 *  26 ± 16 3.0 mg/kg (n = 5) 79 ± 12 * 78 ± 10 * 20± 11 * 30 ± 12 *^(, #) The data represents average values ± standarddeviation * Represent values statistically higher than control, p < 0.05versus control. ^(#) Represent values statistically higher than ExtraLow Dose, p < 0.05 versus Extra Low Dose. ^(&) Represent valuesstatistically higher than Lowered Low Dose, p < 0.05 versus Lowered LowDose.

TABLE 3 Five weeks post-fracture mechanical testing with Vanadium (VAC)in normal rats Fractured femur values Maximum Maximum Torque toTorsional Rigidity Effective Shear Effective Shear failure (Nnun)(Nmm²/rad) Modulus (MPa) Stress (MPa) Control (n = 6) 295 ± 164 20,111 ±10,944 1,060 ± 693 45 ± 28 1.5 mg/kg VAC (n = 9) 471 ± 91 *^(, #) 34,522± 8,347 *  2,026 ± 924 75 ± 26 3.0 mg/kg (n = 8) 335 ± 89  37,496 ±12,846 * 1,453 ± 683 43 ± 25 Fractured femur values normalized to thecontralateral (intact) femur Percent maximum Percent torque to Percentmaximum Percent Effective Effective Shear failure torsional rigidityShear Modulus Stress Control (n = 6) 74 ± 42 80 ± 57 28 ± 29 31 ± 21 1.5mg/kg VAC (n = 9) 99 ± 17 103 ± 33  39 ± 26 47 ± 25 3.0 mg/kg (n = 8) 64± 26 98 ± 28 23 ± 9  22 ± 11 The data represents average values ±standard deviation * Represent values statistically higher than control,p < 0.05 versus control. ^(#) Represent values statistically higher thanhigh dose, p < 0.05 versus high dose.

The results of four to five weeks post-fracture mechanical testing withVAC, in conjunction with CaSO₄ or alone, are listed in Table 2, Tables3, and Table 4, respectively.

TABLE 4 Four weeks post-fracture mechanical testing with VAC and CaSO₄in normal rats Maximum Maximum Torque to Torsional Rigidity EffectiveShear Effective Shear failure (Nmm) (Nmm²/rad) Modulus (MPa) Stress(MPa) Control (n = 6) 161 ± 48  9,889 ± 4,719 258 ± 108 17 ± 4  CaSO₄Buffer (n = 9) 241 ± 172 25,684 ± 20,795 680 ± 623 23 ± 16 0.25 mg/kgVAC and 430 ± 133 * 31,138 ± 11,518 1,178 ± 484 * 55 ± 21*^(, #, &)CaSO₄ Carrier (n = 6) 1.5 mg/kg VAC and 322 ± 157 26,302 ± 17,974 637 ±395 29 ± 15 CaSO₄ Carrier (n = 5) Fractured femur values normalized tothe contralateral (intact) femur Percent maximum Percent torque toPercent maximum Percent Effective Effective Shear failure torsionalrigidity Shear Modulus Stress Control (n = 6) 27 ± 18 20 ± 10 4 ± 2 10 ±5 CaSO₄ Buffer (n = 9) 37 ± 30 47 ± 47 7 ± 7  9 ± 6 0.25 mg/kg VAC and85 ± 24 *^(, #) 100 ± 49* 24 ± 10 *^(, #) 18 ± 9 CaSO₄ Carrier (n = 6)1.5 mg/kg VAC and 64 ± 30 69 ± 47 15 ± 8  10 ± 7 CaSO₄ Carrier (n = 5)The data represents average values ± standard deviation * Representvalues statistically higher than control, p < 0.05 versus control. ^(#)Represent values statistically higher than CaSO₄ Buffer , p < 0.05versus CaSO₄ Buffer. Represent values statistically higher than Low Doseand CaSO₄ Carrier, p < 0.05 versus Low Dose and CaSO₄ Carrier.

The insulin mimetic strategy of the present invention is furtherillustrated by comparison of the mechanical testing parameters of bonefracture healing of the femur in treatment of normal and diabeticsubjects (Table 5), which shows that the 6-week values of the mechanicalparameters are very low for the diabetic subjects but when the VAC wasused at low dosages, these values increased substantially to valuesobserved in normal subject fracture healing and even exceeded the valuesof a bone fracture for a normal subject after the same period of time.

TABLE 5 Comparison of the mechanical testing parameters of bone fracturehealing of the femur in normal, diabetic and diabetic rats treated withVAC/carrier after 6 weeks. Maximum Torque Maximum Torsional EffectiveShear to failure (Nmm) Rigidity (Nmm²/rad) Stress (MPa) diabetic control(n = 23)^(†) 154 ± 69 425 ± 259 3 ± 2 1.5 mg/kg VAC in diabetic 410 ±71 * 43,089 ± 19,720 * 98 ± 53 *^(, #) (n = 3) normal (n = 12) 456 ±66 * 33,784 ± 11,849 * 48 ± 16 * Normalized to Percent maximum Percentmaximum Percent Effective contralateral femur torque to failuretorsional rigidity Shear Stress diabetic control (n = 23) 27 ± 10 27 ±15 8 ± 4 1.5 mg/kg VAC in diabetic 85 ± 23 * 136 ± 111 * 33 ± 20 * (n =3) normal (n = 12) 78 ± 15 * 86 ± 29 28 ± 13 * The data representsaverage values ± standard deviation * Represent values statisticallyhigher than control, p < 0.05 versus control. ^(#) Represent valuesstatistically higher than normal, p < 0.05 versus normal. ^(†)The valueof the numbers for diabetic control is obtained from three papers ofGandhi (Insulin: Bone 2005; PRP:Bone 2006 and Beam et al 2002 JOR). Thevalues of 6 week normal group is an average of Gandhi's paper and DavePaglia 6 week mechanical test saline animals.

The above data has indicated that local vanadium treatment is aneffective method to treat fracture patients, in particular non-diabeticpatients. Mechanical parameters and microradiography revealeddemonstrated that bone has bridged at 4 weeks post fracture. Spiralfractures that occurred during mechanical testing reaffirm thisphenomenon and suggest that local VAC application at the dosages tested,without a carrier may heal bone more than twice as rapidly as salinecontrols. This evidence opens up many future applications to the use ofVAC alone, or incorporated with a carrier as an option for fracturehealing.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims.

1-30. (canceled)
 31. A bone injury treatment kit comprising acomposition sterilized for in vivo bone administration comprising aninsulin-mimetic vanadium compound and a pharmaceutically suitablecarrier for localized bone delivery, wherein said composition ispackaged in a container for localized administration of said vanadiumcompound to a patient in need thereof.
 32. The bone injury treatment kitof claim 31, wherein said vanadium compound is selected from the groupconsisting of vanadyl acetylacetonate (VAC), vanadyl sulfate (VS),vanadyl 3-ethylacetylacetonate (VET), and bis(maholato)oxovanadium(BMOV).
 33. The bone injury treatment kit of claim 31, wherein saidvanadium compound is vanadyl acetylacetonate (VAC).
 34. The bone injurytreatment kit of claim 31, further comprising a bone graft biocomposite.35. The bone injury treatment kit of claim 31, further comprising abioactive agent for treating injured bone tissue.
 36. The bone injurytreatment kit of claim 34, wherein the bone graft biocomposite isselected from the group consisting of allografts, xenografts,alloplastic grafts and synthetic grafts.
 37. The bone injury treatmentkit of claim 35, wherein said bioactive agent is selected from the groupconsisting of peptide growth factors, anti-inflammatory factors,pro-inflammatory factors, inhibitors of apoptosis, MMP inhibitors andbone catabolic antagonists.
 38. The bone injury treatment kit of claim37, wherein said peptide growth factor is selected from the groupconsisting of IGF (1, 2), PDGF (AA, AB, BB), BMPs, FGF (1-20), TGF-beta(1-3), aFGF, bFGF, EGF, VEGF, parathyroid hormone (PTH), and parathyroidhormone-related protein (PTHrP).
 39. The bone injury treatment kit ofclaim 37, wherein said anti-inflammatory factor is selected from thegroup consisting of anti-TNFα, soluble TNF receptors, IL1ra, soluble IL1receptors, IL4, IL-10, and IL-13.
 40. The bone injury treatment kit ofclaim 37, wherein said bone catabolic antagonist is selected from thegroup consisting’ of bisphosphonates, osteoprotegerin, and statins. 41.The bone injury treatment kit of claim 31, wherein the composition isformulated for a human patient.
 42. The bone injury treatment kit ofclaim 41, wherein the composition is formulated for a non-diabetic humanpatient.
 43. The bone injury treatment kit of claim 31, wherein thecomposition is formulated for administration to an animal selected fromthe group consisting of horses, dogs and cats.